Apr 12, 2024
3,3-Diaminobenzidine Tetrahydrochloride (DAB) Immunohistochemistry on Mouse Brain Tissue Sections
- 1Van Andel Research Institute
Protocol Citation: madalynn.erb Erb 2024. 3,3-Diaminobenzidine Tetrahydrochloride (DAB) Immunohistochemistry on Mouse Brain Tissue Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl497zjgo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 08, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 98141
Keywords: ASAPCRN
Abstract
This protocol details the staining for free floating mouse brain sections.
Materials
Pyrex® Staining DishTed Pella Inc.Catalog #36754-60
8-Section Staining NetsTed Pella Inc.Catalog #36154-64
VECTASTAIN® Elite® ABC-HRP Kit, Peroxidase (Standard)Vector LaboratoriesCatalog #PK-6100
Pierce™ DAB Substrate KitThermo ScientificCatalog #34002
Fisherbrand™ Superfrost™ Plus Microscope SlidesFisher ScientificCatalog #12-550-15
EntellanElectron Microscopy SciencesCatalog #14802
Recipes
10X TBS
| A | B | |
| 1M Tris | 500 ml | |
| 5M NaCl | 300 ml | |
| H2O | 200 mL |
1X TBS + 0.1% triton → 1 liter
| A | B | |
| 10X TBS | 100 ml | |
| Triton X | 1 ml | |
| Water | 899 ml |
TBS → 1 liter
| A | B | |
| 1M Tris | 50 ml | |
| 5M NaCl | 30ml | |
| Water | 919 ml |
TBST (0.1% Tween-20) → 1 liter
| A | B | |
| 10X TBS | 100 ml | |
| Tween-20 | 1ml | |
| Water | 899 ml |
0.2M Phosphate Buffer
| A | B | |
| Na2HPO4 | 22.72 g | |
| NaH2PO4 | 5.52 g | |
| H2O | 1000 mL |
0.1M Phosphate Buffer
| A | B | |
| 0.2M PB | 500 mL | |
| H2O | 500 mL |
Cresyl Violet (500 mL)
| A | B | |
| H2O | 500 mL | |
| Cresyl Violet Acetate | 0.5g | |
| 100% Glacial Acetic Acid | 1.25mL |
Warm to 60°C and stir until completely dissolved filter with whatman paper (wrap in foil to protect from light)
Quenching solution→100ml
| A | B | |
| 30% hydrogen peroxide (Sigma H1009) | 1 ml | |
| Methanol | 99 ml |
ABC mix→60ml
| A | B | |
| TBS | 60 ml | |
| Reagent A | 24 drops | |
| Reagent B | 24 drops |
DAB Solution→30ml
| A | B | |
| PBS | 30 ml | |
| (DAB reagent 1) | 12 drops buffer pH = 7.5 | |
| (DAB reagent 2) | 24 drops DAB | |
| (DAB reagent 3) | 12 drops hydrogen peroxide |
vortex between reagents
Blocking solution→100ml
| A | B | |
| NGS | 10 ml | |
| TBS + triton | 90 ml + 0.1% |
Primary Antibodies:
| A | B | C | D | |
| Target | Species | Conc | Manufacturer | |
| Iba1 | Rabbit | 1:1000 | WAKO 019-19741 | |
| GFAP | Mouse | 1:1000 | Sigma G3893 | |
| pSer129-Synuclein | Rabbit | 1:1000 | Abcam ab51253 | |
| TH | Rabbit | 1:2000 | Novus Biological N300109 | |
| pSer202, pThr205 Tau (AT8) | Mouse | 1:1000 | Thermofisher MN1020 | |
| GFP | Rabbit | 1:1000 | Thermofisher A-11122 |
Secondary Antibodies:
| A | B | C | D | E | |
| Target | Species | Conc | Manufacturer | ||
| Mouse | Goat | 1:500 | BA-9200-1.5 | Biotinylated | |
| Rabbit | Goat | 1:500 | BA-1000-1.5 | Biotinylated |
Day 1
Day 1
2d 1h 35m
Staining protocol for 35μm free floating mouse brain sections.
Staining is performed in glass staining dishes (Pyrex 36754-60) using 8-section staining nets (Ted Pella 36154-64) .
The sections can be transferred between wells using a paint brush.
- Volume of solution:
- 20 mL -30 mL for antibodies or ABC mix .
- 50 mL for washing.
Gently rock the plates during the washing and incubation steps.
Wash sections: 3 times (5 min per wash) in Tris Buffer Saline (TBS) at Room temperature .
Wash sections: Wash for 00:05:00 in Tris Buffer Saline (TBS) at Room temperature (1/3).
5m
Wash sections: Wash for 00:05:00 in Tris Buffer Saline (TBS) at Room temperature (2/3).
5m
Wash sections: Wash for 00:05:00 in Tris Buffer Saline (TBS) at Room temperature (3/3).
5m
Quenching: Incubate sections in Quenching Solution (0.3% H202 in Methanol) for 00:10:00 at 4 °C .
10m
Wash sections: 3 short rinses in TBS then 2 times 5min in TBS at Room temperature .
Then wash for 00:05:00 in TBS at Room temperature (1/2).
5m
Then wash for 00:05:00 in TBS at Room temperature (2/2).
5m
Block sections in blocking solution (10% Normal Goat Serum (NGS) + 0.1% Triton-X in TBS) for 01:00:00 at Room temperature .
1h
Primary Antibodies: Dilute antibodies in 5% NGS + 0.1% Triton-X in TBS.
Incubate in primary antibody solution for 48:00:00 at 4 °C .
2d
Cover dishes with parafilm for this step.
Day 4
Day 4
1d 3h 47m
Wash 3 times (10 min each wash) in TBST (0.1% Tween 20 in TBS) at Room temperature .
Wash for 00:10:00 in TBST (0.1%Tween 20 in TBS) at Room temperature (1/3).
10m
Wash for 00:10:00 in TBST (0.1%Tween 20 in TBS) at Room temperature (2/3).
10m
Wash for 00:10:00 in TBST (0.1%Tween 20 in TBS) at Room temperature (3/3).
10m
Secondary Antibodies:
Dilute secondary antibodies in TBST.
Biotinylated goat anti-rabbit IgG (1:500 dilution) or Biotinylated goat anti-mouse IgG (1:500 dilution).
Incubate for 02:00:00 atRoom temperature or 24:00:00 at 4 °C .
1d 2h
Wash sections 3 times (10min each wash) in TBST at Room temperature .
Wash for 00:10:00 in TBST at Room temperature (1/3).
10m
Wash for 00:10:00 in TBST at Room temperature (2/3).
10m
Wash for 00:10:00 in TBST at Room temperature (3/3).
10m
Prepare ABC solution using VECTASTAIN® Elite® ABC-HRP Kit, Peroxidase (Standard) (PK-6100).
ABC mix: 5 mL TBS + 2 drops of reagent A (vortex) + 2 drops reagent B (vortex) let stand at Room temperature for 00:30:00 before using.
30m
Vortex between reagents (after adding reagent A and after adding reagent B).
Make 30 mL ABC solution per dish.
Incubate sections in ABC solution for 01:00:00 at Room temperature .
1h
Wash sections 3 times (10min each wash) in TBST at Room temperature .
Wash for 00:10:00 in TBST at Room temperature (1/3).
10m
Wash for 00:10:00 in TBST at Room temperature (2/3).
10m
Wash for 00:10:00 in TBST at Room temperature (3/3).
10m
Wash sections 2 times (5 min each wash) in TBS at Room temperature .
Wash sections for 00:05:00 in TBS at Room temperature (1/2).
5m
Wash sections for 00:05:00 in TBS at Room temperature (2/2).
5m
Prepare DAB solution using DAB Substrate Kit, Peroxidase (HRP), with Nickel, (3,3'-diaminobenzidine) (SK-4100).
- 5 mL TBS + 2 drops of Reagent 1 (Buffer pH=7.5) + 4 drops Reagent 2 (DAB) + 2 drops Reagent 3 (H2O2).
- Vortex between reagents (after reagent 1, after reagent 2 and after reagent 3).
- Make 30 mL DAB solution per dish.
Revelation with DAB chromogen (until cells are visible):
Note
typically about 00:02:00 for TH.
- All dishes must be developed for the same amount of time – do them together, ask a friend for help if you have more than 2 dishes of tissue.
2m
Perform 3 short rinses in TBS at Room temperature .
Perform rinse in TBS at Room temperature (1/3).
Perform rinse in TBS at Room temperature (2/3).
Perform rinse in TBS at Room temperature (3/3).
Wash sections 5 time (5 min each wash) in TBS at Room temperature .
Wash sections for 00:05:00 in TBS at Room temperature (1/5).
5m
Wash sections for 00:05:00 in TBS at Room temperature (2/5).
5m
Wash sections for 00:05:00 in TBS at Room temperature (3/5).
5m
Wash sections for 00:05:00 in TBS at Room temperature (4/5).
5m
Wash sections for 00:05:00 in TBS at Room temperature (5/5).
5m
Mount sections:
Use a petri dish filled with PBS to mount the sections on superfrost plus slides (Fisher Scientific 12-550-15).
- This is easiest to do using a medium sized paint brush.
After mounting let slides dry at Room temperature (00:02:00 -00:10:00 ) then dip in miliQ H2O.
12m
Dry slides Overnight at Room temperature before moving to the next step.
5m
Day 5 or 6
Day 5 or 6
9h 18m
Rehydrate slides in H2O for 00:02:00 -00:03:00 at Room temperature .
5m
Dehydrate slides in sequential EtOH washes (70%, 95%, 100% EtOH) for 00:05:00 each wash at Room temperature .
5m
Clear tissue for 00:05:00 in fresh Xylene I then incubate in fresh Xylene for ≥ 00:05:00 .
a. Keep slides in Xylene until coversliping.
b. Perform Xylene steps and coversliping in a chemical fume hood.
10m
Use Entellan mounting medium (Electron Microscopy Sciences 14802) to coverslip slides.
Apply one line of Entellan across the middle of the slide.
Cover with glass coverslip and gently press down with forceps.
Let slides dry in the fume hood for 01:00:00 – then dry on the bench Overnight at Room temperature .
1h 5m
Gently remove excess Entellen with a razor blade.
Optional Cresyl Violet Protocol (NISSL stain)
Optional Cresyl Violet Protocol (NISSL stain)
10m
After rehydration and before ethanol steps (dehydration).
Incubate slides in fresh cresyl violet for 00:10:00 at Room temperature .
10m
Proceed with dehydration steps normally.
Must stain with cresyl violet for TH stereology in substantia nigra.
Secondary Antibodies
Secondary Antibodies
| A | B | C | D | E | |
| Target | Species | Conc | Manufacturer | ||
| Mouse | Goat | 1:500 | BA-9200-1.5 | Biotinylated | |
| Rabbit | Goat | 1:500 | BA-1000-1.5 | Biotinylated |